S. cerevisiae is a prominent model organism and a highly valued chassis in the field of synthetic biology. In this space, metabolic engineering is a major focus, as the expression of one or more heterologous enzymes can transform S. cerevisiae into a tiny cellular factory. The most well-known example of this to date is the engineering of S. cerevisiae to produce commercially relevant concentrations of artemisinic acid, a precursor to the anti-malarial drug artemisinin. These metabolic engineering projects require both the introduction of heterologous genes whose expression levels are finely tuned, and the redirection of endogenous biosynthetic pathways via modification of native genes. The development of tools to aid in construction and manipulation of both native and non-native genes for expression in S. cerevisiae thus facilitates metabolic engineering and synthetic biology in yeast.
Typical yeast protein coding genes have a relatively simple anatomy, due in part to the compact structure of the S. cerevisiae genome. Promoters are short, generally extending only ˜500 bp upstream of the start codon. Only ˜20% of promoters in the yeast genome contain TATA boxes. On average, native coding sequences (CDS) are ˜1 kb long and less than 5% contain introns. Sequences associated with 3′ end formation, which typically extend ˜200 bp downstream of the stop codon, are usually AT-rich and contain information for both transcriptional termination and 3′ end processing. The simple structure of yeast genes means that expression of non-native proteins in yeast can be achieved by encoding the CDS of interest between a promoter and terminator that can function in S. cerevisiae. Tuning of CDS expression level can then be accomplished by varying the promoter and terminator sequences, changing the gene copy number (e.g. high or low copy plasmid), or altering the genomic locus in which the gene is integrated.
The production of high-value metabolites in microorganisms suited to industrial scale growth can overcome costly issues associated with traditional production routes, including yield, extraction, or complicated synthesis procedures. To achieve this, the biosynthetic pathway of interest must be re-constructed in an appropriate host organism, typically chosen because it is well characterized and genetically tractable. Saccharomyces cerevisiae is a favored eukaryotic microorganism for metabolic engineering because it is industrially robust, generally regarded as safe, and highly amenable to and tolerant of genetic manipulation. Many recent successes in the metabolic engineering of S. cerevisiae have been described, most notably the cost-effective production of artemisinic acid, a precursor to the anti-malarial drug artemisinin. Engineering of the host genome to redirect endogenous pathways and optimizing the expression levels of non-native biosynthetic genes are keys to successful metabolic engineering projects. However, there remain significant challenges to efficiently assembling biosynthetic pathways and other gene sets for expression in S. cerevisiae. The present disclosure meets these and other challenges.